Effect of TRPV4 Antagonist GSK2798745 on Chlorine Gas-Induced Acute Lung Injury in a Swine Model.

As a regulator of alveolo-capillary barrier integrity, Transient Receptor Potential Vanilloid 4 (TRPV4) antagonism represents a promising strategy for reducing pulmonary edema secondary to chemical inhalation. In an experimental model of acute lung injury induced by exposure of anesthetized swine to chlorine gas by mechanical ventilation, the dose-dependent effects of TRPV4 inhibitor GSK2798745 were evaluated. Pulmonary function and oxygenation were measured hourly; airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, and histopathology were assessed 24 h post-exposure. Exposure to 240 parts per million (ppm) chlorine gas for ≥50 min resulted in acute lung injury characterized by sustained changes in the ratio of partial pressure of oxygen in arterial blood to the fraction of inspiratory oxygen concentration (PaO2/FiO2), oxygenation index, peak inspiratory pressure, dynamic lung compliance, and respiratory system resistance over 24 h. Chlorine exposure also heightened airway response to methacholine and increased wet-to-dry lung weight ratios at 24 h. Following 55-min chlorine gas exposure, GSK2798745 marginally improved PaO2/FiO2, but did not impact lung function, airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, or histopathology. In summary, in this swine model of chlorine gas-induced acute lung injury, GSK2798745 did not demonstrate a clinically relevant improvement of key disease endpoints.

The Costs of Creatinine Testing in the Context of a HIV Pre-Exposure Prophylaxis Demonstration Project in Eswatini.

HIV treatment and prevention as well as other chronic disease care can require regular kidney function assessment based on a creatinine test. To assess the costs of creatinine testing in a public health care system, we conducted activity-based costing during a HIV pre-exposure prophylaxis (PrEP) demonstration project in the Hhohho region of Eswatini. Resource use was assessed by a laboratory technician and valued with government procurement prices, public sector salaries, and own cost estimates. Obtaining a blood sample in a clinic and performing a creatinine test in a high-throughput referral laboratory (> 660,000 blood tests, including > 120,000 creatinine tests, in 2018) were estimated to have cost, on average, $1.98 in 2018. Per test, $1.95 were variable costs ($1.38 personnel, ¢39 consumables, and ¢18 other costs) and ¢2.6 were allocated semi-fixed costs (¢1.1 laboratory equipment, ¢0.85 other, ¢0.45 consumables, and ¢1.3 personnel costs). Simulating different utilization of the laboratory indicated that semi-fixed costs of the laboratory (e.g., equipment purchase or daily calibration of the chemistry analyzer) contributed less than variable costs (e.g., per-test personnel time and test reagents) to the average creatinine test cost when certain minimum test numbers can be maintained. Our findings suggest, first, lower creatinine testing costs than previously used in cost and cost-effectiveness analyses of HIV services and, second, that investment in laboratory equipment imposed a relatively small additional cost on each performed test in the high-throughput referral laboratory.

RESUMEN: El tratamiento y la prevención del VIH, así como el cuidado de otras enfermedades crónicas, pueden requerir una evaluación periódica de la función renal basada en una prueba de creatinina. Para evaluar los costes de las pruebas de creatinina en un sistema de atención sanitaria público, realizamos un cálculo de costes basado en actividades durante un proyecto de demostración de profilaxis preexposición al VIH (PrEP) en la región de Hhohho de Eswatini. El uso de los recursos fue evaluado por un técnico de laboratorio y valorado con los precios de adquisición del gobierno, los salarios del sector público y las estimaciones de costes propias. La obtención de una muestra de sangre en una clínica y la realización de una prueba de creatinina en un laboratorio de referencia de alto rendimiento (> 660.000 pruebas de sangre, incluidas > 120.000 pruebas de creatinina, en 2018) se estimó que habían costado, en promedio, $1,98 en 2018. Por prueba, $1,95 eran costes variables ($1,38 de personal, ¢39 de consumibles y ¢18 de otros costes) y ¢2,6 eran costes semifijos asignados (¢1,1 de equipamiento de laboratorio, ¢0,85 de otros, ¢0,45 de consumibles y ¢1,3 de personal). La simulación de utilización diferente del laboratorio indicó que los costes semifijos del laboratorio (por ejemplo, la compra de equipos o la calibración diaria del analizador químico) contribuyeron menos que los costes variables (por ejemplo, el tiempo del personal por prueba y los reactivos de la prueba) al coste medio de la prueba de creatinina cuando se pueden mantener ciertos números mínimos de pruebas. Nuestros resultados sugieren, en primer lugar, que los costes de las pruebas de creatinina son inferiores a los utilizados anteriormente en los análisis de coste y costo-efectividad de los servicios de VIH y, en segundo lugar, que la inversión en equipos de laboratorio supuso un coste adicional relativamente pequeño en cada prueba realizada en el laboratorio de referencia de alto rendimiento.

Evaluation of GSK2789917-induced TRPV4 inhibition in animal models of fluid induced lung injury.

Administration of bolus intravenous fluids, common in pre-hospital and hospitalised patients, is associated with increased lung vascular permeability and mortality outside underlying disease states. In our laboratory, the induction of lung injury and oedema through rapid administration of intravenous fluid in rats was reduced by a non-specific antagonist of transient receptor potential vanilloid 4 (TRPV4) channels. The aims of this study were to determine the effect of selective TRPV4 inhibition on fluid-induced lung injury (FILI) and compare the potency of FILI inhibition to that of an established model of TRPV4 agonist-induced lung oedema. In a series of experiments, rats received specific TRPV4 inhibitor (GSK2789917) at high (15 µg/kg), medium (5 µg/kg) or low (2 µg/kg) dose or vehicle prior to induction of lung injury by intravenous infusion of TRPV4 agonist (GSK1016790) or saline. GSK1016790 significantly increased lung wet weight/body weight ratio by 96% and lung wet-to-dry weight ratio by 43% in vehicle pre-treated rats, which was inhibited by GSK2789917 in a dose-dependent manner (IC50 = 3 ng/mL). Similarly, in a single-dose study, bolus saline infusion significantly increased lung wet weight/body weight by 17% and lung wet-to-dry weight ratio by 15%, which was attenuated by high dose GSK2789917. However, in a final GSK2789917 dose-response study, inhibition did not reach significance and an inhibitory potency was not determined due to the lack of a clear dose-response. In the FILI model, TRPV4 may have a role in lung injury induced by rapid-fluid infusion, indicated by inconsistent amelioration with high dose TRPV4 antagonist.

Serial MRI characterization of the functional and morphological changes in mouse lung in response to cardiac remodeling following myocardial infarction.

The temporal evolution of heart failure and associated pulmonary congestion in rodent heart failure models has not yet been characterized simultaneously and noninvasively. In this study, MRI was used to assess the serial progression of left-ventricular dysfunction and lung congestion in mice following myocardial infarction (MI). Cardiac and lung (1) H MRI was performed at baseline and every 3 days up to 13 days postsurgery in sham and MI mice. Respiratory parameters and terminal lung mechanics were assessed followed by histological analysis. MRI revealed that the MI induced significant pulmonary congestion/edema as detected by increased MRI signal intensity and was associated with increased lung volume and reduced cardiac contractility. Pulmonary function was also depressed in MI-mice, reflected by a reduced tidal volume and a low minute ventilation rate. Additionally, MI significantly increased lung resistance, markedly reduced lung compliance and total lung capacity and significantly increased lung weights by 57%. Significant correlations were observed between the MRI measured lung congestion, lung volume, ejection fraction, and lung wet-weight parameters. This study demonstrates that MRI may be of significant value in evaluating therapies aimed at primary intervention for lung congestion and secondary prevention of unfavorable cardiac remodeling.

Preclinical evaluation of EPHX2 inhibition as a novel treatment for inflammatory bowel disease.

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by cytochrome P450 epoxygenation of arachidonic acid, which are metabolized by EPHX2 (epoxide hydrolase 2, alias soluble epoxide hydrolase or sEH). EETs have pleiotropic effects, including anti-inflammatory activity. Using a Connectivity Map (CMAP) approach, we identified an inverse-correlation between an exemplar EPHX2 inhibitor (EPHX2i) compound response and an inflammatory bowel disease patient-derived signature. To validate the gene-disease link, we tested a pre-clinical tool EPHX2i (GSK1910364) in a mouse disease model, where it showed improved outcomes comparable to or better than the positive control Cyclosporin A. Up-regulation of cytoprotective genes and down-regulation of proinflammatory cytokine production were observed in colon samples obtained from EPHX2i-treated mice. Follow-up immunohistochemistry analysis verified the presence of EPHX2 protein in infiltrated immune cells from Crohn's patient tissue biopsies. We further demonstrated that GSK2256294, a clinical EPHX2i, reduced the production of IL2, IL12p70, IL10 and TNFα in both ulcerative colitis and Crohn's disease patient-derived explant cultures. Interestingly, GSK2256294 reduced IL4 and IFNγ in ulcerative colitis, and IL1ß in Crohn's disease specifically, suggesting potential differential effects of GSK2256294 in these two diseases. Taken together, these findings suggest a novel therapeutic use of EPHX2 inhibition for IBD.

Tumor-specific efficacy of transforming growth factor-beta RI inhibition in Eker rats.

PURPOSE: Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions. EXPERIMENTAL DESIGN: We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency. RESULTS: Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals. CONCLUSION: Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors.

Atherosclerotic plaque inflammation varies between vascular sites and correlates with response to inhibition of lipoprotein-associated phospholipase A2.

BACKGROUND: Despite systemic exposure to risk factors, the circulatory system develops varying patterns of atherosclerosis for unclear reasons. In a porcine model, we investigated the relationship between site-specific lesion development and inflammatory pathways involved in the coronary arteries (CORs) and distal abdominal aortas (AAs). METHODS AND RESULTS: Diabetes mellitus (DM) and hypercholesterolemia (HC) were induced in 37 pigs with 3 healthy controls. Site-specific plaque development was studied by comparing plaque severity, macrophage infiltration, and inflammatory gene expression between CORs and AAs of 17 DM/HC pigs. To assess the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in plaque development, 20 DM/HC pigs were treated with the Lp-PLA2 inhibitor darapladib and compared with the 17 DM/HC untreated pigs. DM/HC caused site-specific differences in plaque severity. In the AAs, normalized plaque area was 4.4-fold higher (P<0.001) and there were more fibroatheromas (9 of the 17 animals had a fibroatheroma in the AA and not the COR, P=0.004), while normalized macrophage staining area was 1.5-fold higher (P=0.011) compared with CORs. DM/HC caused differential expression of 8 of 87 atherosclerotic genes studied, including 3 important in inflammation with higher expression in the CORs. Darapladib-induced attenuation of normalized plaque area was site-specific, as CORs responded 2.9-fold more than AAs (P=0.045). CONCLUSIONS: While plaque severity was worse in the AAs, inflammatory genes and inflammatory pathways that use Lp-PLA2 were more important in the CORs. Our results suggest fundamental differences in inflammation between vascular sites, an important finding for the development of novel anti-inflammatory therapeutics.

Examining the relationship between exercise tolerance and isoproterenol-based cardiac reserve in murine models of heart failure.

The loss of cardiac reserve is, in part, responsible for exercise intolerance in late-stage heart failure (HF). Exercise tolerance testing (ETT) has been performed in mouse models of HF; however, treadmill performance and at-rest cardiac indexes determined by magnetic resonance imaging (MRI) rarely correlate. The present study adopted a stress-MRI technique for comparison with ETT in HF models, using isoproterenol (ISO) to evoke cardiac reserve responses. Male C57BL/6J mice were randomly subjected to myocardial infarction (MI), transverse aortic constriction (TAC), or sham surgery under general anesthesia. Mice underwent serial ETT on a graded treadmill with follow-up ISO stress-MRI. TAC mice showed consistent exercise intolerance, with a 16.2% reduction in peak oxygen consumption vs. sham at 15-wk postsurgery (WPS). MI and sham mice had similar peak oxygen consumption from 7 WPS onward. Time to a respiratory exchange ratio of 1.0 correlated with ETT distance (r = 0.64; P < 0.001). The change in ejection fraction under ISO stress was reduced in HF mice at 4 WPS [10.1 ± 3.9% change (Δ) and 8.9 ± 3.5%Δ in MI and TAC, respectively, compared with 32.0 ± 3.5%Δ in sham; P < 0.001]. However, cardiac reserve differences between surgery groups were not observed at 16 WPS in terms of ejection fraction or cardiac output. In addition, ETT did not correlate with cardiac indexes under ISO stress. In conclusion, ISO stress was unable to reflect consistent differences in ETT between HF and healthy mice, suggesting cardiac-specific indexes are not the sole factors in defining exercise intolerance in mouse HF models.

An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.

Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

Relationship of microparticles to progenitor cells as a measure of vascular health in a diabetic population.

OBJECTIVE: Quantitative measures are needed to identify diabetic patients at higher risk for CV events. Cell-derived microparticles (MPs) are submicron membrane vesicles released from activated cells that are indicative of cell damage. Progenitor cells (PCs) including proangiogenic cells (PACs), often termed endothelial progenitor cells (EPCs), are mediators of reparative capacity. We examined whether the relationship of MPs to PCs/PACs could be used as an improved and clinically feasible index of vascular pathology. METHODS AND RESULTS: Plasma samples were collected from patients with early-stage (ES, Diagnosis < 1 year) and long-term (LT, Diagnosis > 5 years,) Type 2 diabetes and compared with age related healthy subjects (H). PC and MP subtypes were measured by a combination of flow cytometry and ELISA-based methods. The ratio of procoagulant MPs/CD34(+) PCs proved a valuable index to distinguish between subject groups (P = 0.01). This index of compromised vascular function was highest in the LT group despite intensive statin therapy and was more informative than a range of soluble protein biomarkers. CONCLUSIONS: This is the first report of a relationship between MPs and PCs in Type 2 diabetes. This ratio may provide a quantitative and clinically feasible measurement of vascular dysfunction and cardiovascular risk in patients with diabetes. © 2010 International Clinical Cytometry Society.

Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development.

Increased lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA(2) is a causative agent. Here we show that selective inhibition of Lp-PLA(2) with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA(2) activity and reduced lesion lysophosphatidylcholine content. Analysis of coronary gene expression showed that darapladib exerted a general anti-inflammatory action, substantially reducing the expression of 24 genes associated with macrophage and T lymphocyte functioning. Darapladib treatment resulted in a considerable decrease in plaque area and, notably, a markedly reduced necrotic core area and reduced medial destruction, resulting in fewer lesions with an unstable phenotype. These data show that selective inhibition of Lp-PLA(2) inhibits progression to advanced coronary atherosclerotic lesions and confirms a crucial role of vascular inflammation independent from hypercholesterolemia in the development of lesions implicated in the pathogenesis of myocardial infarction and stroke.

Synthetic LXR agonists increase LDL in CETP species.

Liver X receptor (LXR) nuclear receptors regulate the expression of genes involved in whole body cholesterol trafficking, including absorption, excretion, catabolism, and cellular efflux, and possess both anti-inflammatory and antidiabetic actions. Accordingly, LXR is considered an appealing drug target for multiple indications. Synthetic LXR agonists demonstrated inhibition of atherosclerosis progression in murine genetic models; however, these and other studies indicated that their major undesired side effect is an increase of plasma and hepatic triglycerides. A significant impediment to extrapolating results with LXR agonists from mouse to humans is the absence in mice of cholesteryl ester transfer protein, a known LXR target gene, and the upregulation in mice but not humans of cholesterol 7alpha-hydroxylase. To better predict the human response to LXR agonism, two synthetic LXR agonists were examined in hamsters and cynomolgus monkeys. In contrast to previously published results in mice, neither LXR agonist increased HDL-cholesterol in hamsters, and similar results were obtained in cynomolgus monkeys. Importantly, in both species, LXR agonists increased LDL-cholesterol, an unfavorable effect not apparent from earlier murine studies. These results reveal additional problems associated with current synthetic LXR agonists and emphasize the importance of profiling compounds in preclinical species with a more human-like LXR response and lipoprotein metabolism.